RhoB differentially controls Akt function in tumor cells and stromal endothelial cells during breast tumorigenesis

Abstract

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.

Figure 1

RhoB is expressed in blood vessels and cancer cells in human breast tumors. Antisense probe hybridized to noncancerous mammary duct (A), noncancerous blood vessel in adjacent normal tissue (B), ductal carcinoma (C), and blood vessels in tumor (D). The result shows less expression of RhoB in adjacent normal tissue compared with ductal carcinoma (A vs. C) and less in adjacent normal stromal blood vessels compared with blood vessels in the tumor (B vs. D). The control sense probe on tumor tissue (E) and tumor blood vessel (F) are also shown. Magnification 400×.

Figure 2

RhoB loss promotes the growth of breast tumor cells. A, mammary glands from 8-week-old mice are shown (a) and mean number of lesions graphed (b). n, the number of fat pads that were stained. B, 3D culture of primary tumor cells at 16 days (a). Acini size was quantified by ImageJ software (b). C, tumor cells counted from 0 to 72 hours on polyHEMA-coated dishes. D, BrdUrd incorporation for 48 hours of incubation for RhoB^+/+ and RhoB^−/− cells in 5% serum. See also Supplementary Fig. S1. P values were calculated with Prism4 by an unpaired 2-tailed test. Data are represented as mean ± SEM.

Figure 3

Loss of RhoB in human breast cancer cells promotes hyperproliferation phenotype. A, 3D growth of MDA-MB-231 RhoB silenced (shRhoB) or control (pLKO) breast cancer cells after 2 to 15 days. B, these cells were also assessed for Transwell migration after 4 hours. C, MTT proliferation/survival assays at 24 and 48 hours. D, growth in soft agar (a) with quantitation of 25 colonies per condition [volume = ((4/3)(pi)(rˆ3); (b)]. Data are represented as mean ± SEM. P values were calculated using an unpaired 2-tailed test. See also Supplementary Fig. S2.

Figure 4

RhoB loss in breast tumor cells increases phospho-S473 on all Akt isoforms. A, Western blot analysis on tumor lysates. B, primary tumor cells starved and stimulated with 100 ng/mL insulin-like growth factor (IGF) or EGF for 10 minutes. C, human MDA-MB-231 starved and stimulated with EGF (20 ng/mL for 10 minutes). D, immunoprecipitation-Western blot analysis of phospho-S473 blotted for Akt isoforms 1, 2, and 3, with pAkt and tubulin Western blotting as lysate controls. E and F, RhoB^+/+ and RhoB^−/− mouse tumor cells silenced for the Akt isoforms indicated 3D Matrigel/collagen matrix after 16 days. Acini size was quantified using ImageJ and Prism4 software. See also Supplementary Figs. S2, S4, and S5.

Figure 5

RhoB deficiency delays tumor growth and reduces blood vessel density. A, the percentage of animals with palpable tumor in RhoB^+/+, ^+/−, and ^−/− tumors over time. B, total tumor weight at day 110 for all genotypes. C, representative endothelial (CD31, green) and pericyte (SMA, red) staining of vasculature are shown at magnification 200×. D, vascular content was quantified by real-time PCR of the endothelial-specific gene ve-cadherin (CDH5) relative to GAPDH. E, primary RhoB^+/+, ^+/−, and ^−/− tumor endothelial cells analyzed by immunoblotting for pAkt (S473). F, qRT-PCR quantitation of Akt isoforms in endothelial cells normalized to 1 million copies of 18S RNA.

Figure 6

Positive impact of RhoB on tumor angiogenesis supercedes its negative impact in cancer cells. A, minced RhoB^+/+ tumors were orthotopically transplanted into RhoB^+/− or RhoB^−/− SCID mouse hosts (n = 10). Labels indicate the genotype of the recipient host. B, tumor cells isolated from RhoB^+/− or RhoB^−/− mice in Matrigel were similarly orthotopically transplanted into wild-type SCID mice (n = 10). Labels indicate the genotype of the donor tumor cells. C, human MDA-MB-231 cells were implanted orthotopically into wild-type SCID mice (n = 10). Labels indicate the genotype of the donor tumor cells. D, tumors from B above were stained with anti-CD31 to assess the microvascular density. Representative images are shown. E, mRNA expression levels for murine VEGF-A in RhoB^+/− and RhoB^−/− endothelial cells isolated from tumors were assessed by qRT-PCR.

Figure 7

Loss of RhoB in mouse tumor cells increases EGFR cell surface levels. A, Western blot for EGFR in RhoB^+/+ and RhoB^−/− mouse tumor cells. B, qRT-PCR of EGFR mRNA. C, FACS analysis of EGFR on the cell surface of mouse tumor cells. D, p-EGFR (p-Try1068) and p-Akt (p-S473) after treatment with EGFR-neutralizing antibody ME-1. E, proliferation of mouse tumor cells via BrdUrd incorporation after 72-hour treatment with ME1 in 5% FBS. F, 3D growth in Matrigel culture after ME1 blockade of EGFR signaling (a). The average size of the acini (b). Data are represented as mean ± SEM. P values were calculated using an unpaired 2-tailed test.